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__NOTOC__
 
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<partinfo>BBa_K3076100 short</partinfo>
 
<partinfo>BBa_K3076100 short</partinfo>
  
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==Description==
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<p><br>Metallothioneins (MT) are proteins made of 61-68 amino acids with a small molecular weight. It is found in almost every known organism, from bacteria to humans. Due to this ubiquity, it minimizes the gene toxicity effect when we choose it as the ectopic expression target.</br></p>
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This part contains the coding sequence of <i>Corynebacterium glutamicum</i> metallothionein gene (<i>CgMT</i>). This part was synthesized into pET151 expression vector as an expression construct <html>(<a href="https://parts.igem.org/Part:BBa_K3076803">BBa K3076803</a>)</html> and then transformed into BL21(DE) <i>E. coli</i> strain. The expression of this part showed a significant increase of copper absorption ability in <i>E. coli</i> and it suits our purpose of creating a bacterial absorbent of metal pollutants in the liquid medium.
 +
 
 +
 
 +
==Usage and Biology==
 +
Metallothioneins (MT) are proteins made of 61-68 amino acids with a small molecular weight. It is found in almost every known organism, from bacteria to humans. Due to this ubiquity, it minimizes the gene toxicity effect when we choose it as the ectopic expression target.
 +
 
 +
MT contains many cysteine groups which were reported to be responsible for chelating a wide range of metal ions such as cadmium, lead, copper, and mercury, etc. Nevertheless, MT from different species showed different affinity towards different metal ions.
 +
 
 +
Literature reported that MT protein from <i>Corynebacterium glutamicum</i> (CgMT) shows strong binding affinity towards divalent cations, such as Zn2+ and Pb2+. [1] Since our project used Cu2+ as a model for the metal pollutants, we decided to explore the plausibility of using CgMT to increase the metal accumulation ability of <i>E. coli</i>.
 +
 
 +
===Copper removal assay===
 +
The <i>E. coli</i> expressing CgMT was put into copper (II) sulphate-containing medium. The concentration of copper ions in the medium was measured at different time points.
 +
API copper testing reagent was used in this assay. The reagent reacts with copper ions and forms brown colour precipitate. We used a colourimeter to measure the absorbance (440nm) of the reagent mixture and calculate the concentration of copper ion by a standard curve. (Fig. 1)
  
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<p><br>MT contains many cysteine groups which were reported to be responsible for chelating a wide range of metal ions such as cadmium, lead, copper, and mercury, etc. Nevertheless, MT from different species showed different affinity towards different metal ions.</br><p/>
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[[File:Standard curve for absorbance (440nm) against copper concentration.png|400px|thumb|center|Fig. 2 This standard curve was constructed by using standard solutions of copper (II) sulphate. The absorbance (440nm) of the API reagent and standard solution mixture were measured by colourimeter. The linear equation of the standard curve was then used in other assays to convert the absorbance value into concentration.]]
  
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<p><br>Literature reported that MT protein from Corynebacterium glutamicum (CgMT) shows strong binding affinity towards divalent cations, such as Zn2+ and Pb2+. [1] Since our project used Cu2+ as a model for the metal pollutants, we decided to explore the plausibility of using CgMT to increase the metal accumulation ability of E. coli.</br></p>
+
We found that when compared with no IPTG induction control group and empty vector control group, the <i>CgMT</i> gene -expressing group removed significantly more copper ions inside the medium after 4 hours of incubation. (Fig. 2)
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<p><br>Furthermore, Corynebacterium glutamicum belongs to the same order as Mycobacterium tuberculosis. Previous iGEM team Oxford 2016 demonstrated that MT from M. tuberculosis (BBa_K1980002) chelates copper ions in cells effectively, thus we expected CgMT also chelates copper (II) efficiently and it will increase the accumulation of copper ions inside E. coli expressing CgMT.</br></p>
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This result indicates <i>CgMT</i> gene expression can effectively increase the copper absorption ability of <i>E. coli</i> and it serves the aim of our project which we tried to increase the metal pollutant removal ability of <i>E. coli</i>.
  
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<p><br>This part was synthesized into pET151/TOPO vector for the ectopic expression in E. coli BL21. The results showed that the CgMT transformant absorbed significantly more copper ions (~15%) in the culture medium when compared with non-induced control and empty vector control.</br></p>
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[[File:CgMT result.png|400px|thumb|center|Fig 2. The <i>CgMT</i>-expressing group removed ~30% of copper ions in the medium when compared with controls removing only ~12 - 15%.]]
  
 +
===Practical application===
 +
Our team developed a Bacterial Copper Adsorption Device (B-CAD) that can utilize the metal absorption property of the <i>E. coli</i> to remove copper in a fish tank or aquaponic tank. The device mainly relies on the differentially permeable property of the dialysis tubing so that the <i>E. coli</i> can be trapped inside of the tubings whereas the metal ions can pass through the membrane freely.  (Fig. 3 and 4)
  
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<h3>Assay 1: Test for copper absorption ability on <i>E.coli</i> transformed with CgMT</h3>
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[[File:CAD device.png|700px|thumb|center|Fig. 3 The design of the B-CAD.]]
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<h4>Abstract of experiment:</h4>
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[[File:CAD device built.png|700px|thumb|center|Fig. 4 The mini-version of the B-CAD we constructed.]]
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<p>We utilized pET-151/TOPO vector to drive the expression of CgMT coding sequence under T7 promoter inside BL21(DE) E. coli strain. Then, the bacteria transformants will be tested inside copper (II) sulphate added culture medium to measure their copper absorption efficiency along time.
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</p>
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<h4> Protocol:
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We tested the device with actual aquaponic water added with copper (II) sulphate. (Fig. 5) Actual aquaponic water contains the nutrients that <i>E. coli</i> needed and no extra nutrient broth is required for the growth of the bacteria. Therefore, in practical situation, the cost of growing our <i>CgMT</i> expressing <i>E. coli</i> would be minimized.
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<br> 1. Prepare overnight culture of MT BL21(DE) E. coli transformants and pET28a(+) BL21(DE) E. coli empty vector controls (Incubated at 37oC, 80 rpm)</br>
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<br>2. Transfer overnight culture to freshly prepared Luria broth medium with antibiotics (Ampicillin for MT transformants and Kanamycin for empty vector control) in a ratio of 1:50. Then incubate at 37oC, 80 rpm for 2 hours. </br>
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<br>3. Add IPTG into the designated IPTG+ culture and Empty vector control culture to a final concentration of 0.1 mM. Then incubate at 37oC, 80 rpm for 4 hours. </br>
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<br>4. Adjust the concentration of bacterial culture using LB broth and the absorbance of each culture (IPTG+, IPTG- and Empty vector) is normalized to 1.5.</br>
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<br>5. Add 0.5 mL of 100 mg/L copper (II) sulphate solution to each test tube for assay.</br>
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<br>6. Add 4.5 mL of corresponding bacterial culture to each test tube.</br>
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<br>7. The experimental groups are designed according to the table below:</br>
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<br><p><u>Table 1: Time of incubation in 0, 1, 4, 16 hours with engineered bacteria</u></p>
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[[File:Testing device.png|700px|thumb|center|Fig. 5 Testing the copper removal ability by our B-CAD.]]
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    <th rowspan=1></th>
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    <th colspan="4">Time of incubation (Hours)</th>
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    <th rowspan=1><i>(IPTG+)
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4.5 mL CgMT Transformant with IPTG +
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0.5 mL 100 mg/L CuSO4 </i> MT1 gene</th>
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    <td>0 h</td>
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    <td>1 h</td>
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    <td>4 h</td>
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    <td>16 h</td>
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    <th rowspan=1><i>(IPTG-)
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4.5 mL CgMT Transformant +
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0.5 mL 100 mg/L CuSO4</i> MT1 gene</th>
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    <td>0 h</td>
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    <td>1 h</td>
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    <td>4 h</td>
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    <td>16 h</td>
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  </tr>
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  <tr>
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    <th rowspan=1><i>(Empty)
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4.5 mL Empty vector Transformant with IPTG +
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0.5 mL 100 mg/L CuSO4 </i> MT1 gene</th>
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    <td>0 h</td>
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    <td>1 h</td>
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    <td>4 h</td>
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    <td>16 h</td>
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  </tr>
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  <tr>
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    <th rowspan=1><i>(Blank)
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LB medium
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</i> MT1 gene</th>
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    <td>0 h</td>
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    <td>1 h</td>
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    <td>4 h</td>
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    <td>16 h</td>
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  </tr>
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</table>
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The copper removal effects of the B-CAD were studied with different variables. (Fig. 6, 7 and 8) 50 mL of <i>CgMT</i>-expressing <i>E. coli</i> was put into the B-CAD and used to filter 5 L of aquaponic water with copper (II) sulphate added. The results showed that our B-CAD system could remove ~25% and ~38% of the copper inside the water after 24 hours and 48 hours respectively.
  
 +
[[File:B-CAD with transformants.jpeg|400px|thumb|center|Fig. 6 The copper removal effect of B-CAD when hosting different <i>E. coli</i>. The B-CAD system with <i>CgMT</i>-expressing <i>E. coli</i> showed significantly higher efficiency in lowering the copper concentration in the system.]]
 +
[[File:B-CAD on off result.jpeg|400px|thumb|center|Fig. 7 The efficiency of copper removal of <i>CgMT</i>-expressing <i>E. coli</i> when the pump was on with maximum flow rate (~400 ml/min) compared with the pump was off. This shows that the speed of the flow is an important factor determining the copper removal efficiency.]]
 +
[[File:B-CAD conc. result.jpeg|400px|thumb|center|Fig. 8 The efficiency of copper removal of <i>CgMT</i>-expressing <i>E. coli</i> with different initial copper concentration.]]
  
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<br> 8. When the designated time point is reached, 3 mL of each bacterial culture from the group is centrifuged with 6,000 rpm, 1 minute. </br>
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===Future work===
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<br> 9. 2.5 mL of the supernatant is transferred to the cuvette and added with 175 uL of API copper testing reagent. Pipette up and down to make sure the reagent is completely reacted. </br>
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In this project, we also studied the effect of knocking out copper exporters in <i>E. coli</i> to see if the copper absorption in the organism will be increased. We found that by knocking out <i>cusF</i> gene, the KO strain significantly removed more copper in the medium when compared with the control group. <html>(<a href="https://parts.igem.org/Part:BBa_K3076500">BBa K3076500</a>)</html>
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<br> 10. Measure the absorbance of the reaction mixture by colourimeter with 440 nm. Repeat the measurements twice to ensure no further development of colour. </br>
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Thus, in the next step, we will combine two modifications together to see if it can further improve our metaL pollutant removal system.
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<br> 11. Record the absorbance and convert it to concentration value by the standard curve. </br>
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<h3>Results:</h3>
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===References===
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<br><p><u>Graph 1: Percentage decrease of copper concentration along time</u></p>
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[1] Jafarian, V., & Ghaffari, F. (2017). A unique metallothionein-engineered in <i>Escherichia coli</i> for biosorption of lead, zinc, and cadmium; absorption or adsorption? Microbiology, 86(1), 73–81. doi: 10.1134/s0026261717010064
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<img src="https://static.igem.org/mediawiki/parts/1/18/T--Hong_Kong_JSS--result_for_CgMT.png" ><br>
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<p><u>Graph 1: The percentage decrease of copper concentration in the culture medium
 
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In the graph, the percentage change of copper(II) ion concentration along time is shown. The copper added media were incubated with IPTG+ (IPTG induced CgMT expression E. coli), IPTG- (CgMT expression E. coli without IPTG added) and Empty (Empty vector control E. coli). IPTG+ showed ~30% decrease, IPTG- showed ~18% decrease and empty vector control showed ~14% decrease.</u></p>
 
  
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<br>The result shows that IPTG+ (blue) had a significant increase in copper removal ability when compared with IPTG- control (red) and Empty control (green). The purple curve shows the blank control in which no bacteria were inoculated in the copper added medium.</br>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3076100 SequenceAndFeatures</partinfo>
  
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<br>It indicates that CgMT expression contributes to the copper absorption ability of the E. coli strains (BL21).
 
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Although IPTG- also showed a statistical difference when compared with empty vector control, we believe it was due to the small sample size or leaky expression of the T7 promoter. Further investigation is required.
 
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This assay was repeated twice and the error bars represent standard deviation which indicates the distribution of data range.
 
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</br>
 
  
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<h3>Future work: </h3>
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<!-- Uncomment this to enable Functional Parameter display
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<br>Since our assay method is relatively insensitive when compared with using advanced techniques such as mass spectrometry, we are going to collaborate with university labs to seek a more sensitive way of measurement and thus we can generate a model with higher precision.</br>
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===Functional Parameters===
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<br>Secondly, we aim to create an E. coli absorbent of heavy metals so we are going to combine CgMT with metal exporter knockout modification and further increase the absorption ability of E. coli.</br>
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<partinfo>BBa_K3076100 parameters</partinfo>
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</html>
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<!-- -->

Latest revision as of 11:42, 15 October 2019


Coding sequence of metallothioneins (MT) gene from Corynebacterium glutamicum (CgMT)

Description

This part contains the coding sequence of Corynebacterium glutamicum metallothionein gene (CgMT). This part was synthesized into pET151 expression vector as an expression construct (BBa K3076803) and then transformed into BL21(DE) E. coli strain. The expression of this part showed a significant increase of copper absorption ability in E. coli and it suits our purpose of creating a bacterial absorbent of metal pollutants in the liquid medium.


Usage and Biology

Metallothioneins (MT) are proteins made of 61-68 amino acids with a small molecular weight. It is found in almost every known organism, from bacteria to humans. Due to this ubiquity, it minimizes the gene toxicity effect when we choose it as the ectopic expression target.

MT contains many cysteine groups which were reported to be responsible for chelating a wide range of metal ions such as cadmium, lead, copper, and mercury, etc. Nevertheless, MT from different species showed different affinity towards different metal ions.

Literature reported that MT protein from Corynebacterium glutamicum (CgMT) shows strong binding affinity towards divalent cations, such as Zn2+ and Pb2+. [1] Since our project used Cu2+ as a model for the metal pollutants, we decided to explore the plausibility of using CgMT to increase the metal accumulation ability of E. coli.

Copper removal assay

The E. coli expressing CgMT was put into copper (II) sulphate-containing medium. The concentration of copper ions in the medium was measured at different time points. API copper testing reagent was used in this assay. The reagent reacts with copper ions and forms brown colour precipitate. We used a colourimeter to measure the absorbance (440nm) of the reagent mixture and calculate the concentration of copper ion by a standard curve. (Fig. 1)

Fig. 2 This standard curve was constructed by using standard solutions of copper (II) sulphate. The absorbance (440nm) of the API reagent and standard solution mixture were measured by colourimeter. The linear equation of the standard curve was then used in other assays to convert the absorbance value into concentration.

We found that when compared with no IPTG induction control group and empty vector control group, the CgMT gene -expressing group removed significantly more copper ions inside the medium after 4 hours of incubation. (Fig. 2) This result indicates CgMT gene expression can effectively increase the copper absorption ability of E. coli and it serves the aim of our project which we tried to increase the metal pollutant removal ability of E. coli.

Fig 2. The CgMT-expressing group removed ~30% of copper ions in the medium when compared with controls removing only ~12 - 15%.

Practical application

Our team developed a Bacterial Copper Adsorption Device (B-CAD) that can utilize the metal absorption property of the E. coli to remove copper in a fish tank or aquaponic tank. The device mainly relies on the differentially permeable property of the dialysis tubing so that the E. coli can be trapped inside of the tubings whereas the metal ions can pass through the membrane freely. (Fig. 3 and 4)

Fig. 3 The design of the B-CAD.
Fig. 4 The mini-version of the B-CAD we constructed.

We tested the device with actual aquaponic water added with copper (II) sulphate. (Fig. 5) Actual aquaponic water contains the nutrients that E. coli needed and no extra nutrient broth is required for the growth of the bacteria. Therefore, in practical situation, the cost of growing our CgMT expressing E. coli would be minimized.

Fig. 5 Testing the copper removal ability by our B-CAD.

The copper removal effects of the B-CAD were studied with different variables. (Fig. 6, 7 and 8) 50 mL of CgMT-expressing E. coli was put into the B-CAD and used to filter 5 L of aquaponic water with copper (II) sulphate added. The results showed that our B-CAD system could remove ~25% and ~38% of the copper inside the water after 24 hours and 48 hours respectively.

Fig. 6 The copper removal effect of B-CAD when hosting different E. coli. The B-CAD system with CgMT-expressing E. coli showed significantly higher efficiency in lowering the copper concentration in the system.
Fig. 7 The efficiency of copper removal of CgMT-expressing E. coli when the pump was on with maximum flow rate (~400 ml/min) compared with the pump was off. This shows that the speed of the flow is an important factor determining the copper removal efficiency.
Fig. 8 The efficiency of copper removal of CgMT-expressing E. coli with different initial copper concentration.

Future work

In this project, we also studied the effect of knocking out copper exporters in E. coli to see if the copper absorption in the organism will be increased. We found that by knocking out cusF gene, the KO strain significantly removed more copper in the medium when compared with the control group. (BBa K3076500) Thus, in the next step, we will combine two modifications together to see if it can further improve our metaL pollutant removal system.

References

[1] Jafarian, V., & Ghaffari, F. (2017). A unique metallothionein-engineered in Escherichia coli for biosorption of lead, zinc, and cadmium; absorption or adsorption? Microbiology, 86(1), 73–81. doi: 10.1134/s0026261717010064


Sequence and Features


Assembly Compatibility:
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